1. Field of the Invention.
The invention relates to a virus-inactivated blood product, a method of producing the same as well as a method of determining the virus-inactivating capacity of an inactivation treatment.
2. Description of Related Art
By blood products, products from human or animal blood or plasma are meant, which are intended for therapeutic, prophylactic or diagnostic applications. Such products may contain enzymes, proenzymes including coagulation factors, enzyme co-factors, enzyme inhibitors, immunoglobulins, albumin, plasminogen, fibrinogen, fibronectin or plasma.
The administration of blood products involves the risk of infection due to infectious agents possibly present in the donor plasma, such as hepatitis or AIDS viruses. Even if exclusively plasma that has been tested for its absence of these infectious agents is used, the danger that a patient might be infected cannot be excluded because of the limited sensitivity of the test methods. When producing blood products one is therefore forced to inactivate possibly present infectious agents by various methods.
There exists a great number of publications concerned with the inactivation of pathogens in blood products.
The various methods include:
heating the blood products in aqueous solution, possibly with the addition of virucidal substances, PA1 heating the blood products in aqueous solution in the presence of stabilizing agents PA1 treating the blood products with organic solvents and/or detergents, PA1 heating the blood products in dry and wet states, PA1 combined treatment of the blood products with an organic solvent/detergent and heating the blood products in the dry state. PA1 a) the blood product is treated in an aqueous solution containing at least 2%, preferably at least 5%, of a detergent, and subsequently is heated in the solid state, or PA1 b) the blood product is heated in the solid state and subsequently is treated in an aqueous solution containing at least 2%, preferably at least 5%, of a detergent.
All these inactivation methods aim at eliminating the potential infectiousness of the preparations, while maintaining their biological activity as far as possible. However, so far this aim could only be achieved in the case of albumin preparations, by heating aqueous albumin solutions at a temperature of 60.degree. C. for 10 h, because albumin is substantially more stable with regard to the influence of heat than all the other blood proteins.
In detail, the following publications may, e.g., be mentioned to the prior art:
DE-A- 29 16 711 describes a method of treating coagulation-factors-containing preparations in aqueous solution by applying a temperature of from 30.degree. to 100.degree. C., wherein an amino acid or a mono-, oligosaccharide or sugar alcohol are admixed to the solution of the coagulation factors.
EP-A2-0 053 338 describes a method of inactivating hepatitis viruses in preparations containing factors IX and X, wherein the aqueous solution of a blood preparation is heated in the presence of calcium ions and, possibly, an amino acid and/or a saccharide or a sugar alcohol at temperatures of up to 100.degree. C.
In EP-A2-0 035 204 a method of inactivating aqueous protein solutions, which may contain factor VIII, fibronectin, globulin, fibrinogen and other proteins, is disclosed, wherein the composition is mixed with a polyol and the mixture is heated to a temperature of from 60.degree. to 75.degree. C.
In EP-A2-0 052 827 a method of inactivating hepatitis viruses in an aqueous solution containing factors II and VII in the presence of a chelating agent and, possibly, an amino acid and/or a saccharide or sugar alcohol is described.
In U.S. Pat. No. 4,379,085 a method for the thermal inactivation of a plasma protein, such as C.sub.1 -inhibitor or factor IX, in aqueous solution in the presence of potassium or ammonium citrate is disclosed.
In EP-A2-0 077 870 an inactivation method is described, in which an aqueous, factor VIII-containing solution is heated with amino acids, monosaccharides, oligosaccharides, sugar alcohols and hydrocarbon- or hydroxy-hydrocarbon-carboxylic acids having from 3 to 10 carbon atoms, to a temperature of from 50.degree. to 80.degree. C.
In the PCT application WO 83/04371 a method of inactivating hepatitis-viruses is disclosed, wherein a preparation containing the virus is treated at a temperature of from 4.degree. to 40.degree. C. with a halohydrocarbon, in particular chloroform.
EP-B1-0 015 055 discloses a method of treating a blood product, wherein the product is subjected to a microwave radiation treatment in the anhydrous condition so as to inactivate any microorganisms present.
In a treatise of the XII. International Congress on Blood Transfusion, Abstracts, "MIR" Publishers, Moscow 1969, pp. 473-475, Rosenberg et al. disclose a method of inactivating albumin-containing preparations and fibrinogen in the dry state by heating for 10 hours at 60.degree. C.
EP-A2-0 094 611 discloses a method of treating a factor VIII-containing composition in the dry, for instance the lyophilized, state by applying a temperature of at least 60.degree. C. for inactivating any hepatitis viruses present.
The published PCT application WO 82/03871 describes a method of treating preparations containing blood coagulation enzymes, the preparations being heated in the dry state so as to inactivate infectious viruses present; the dry state is defined to be a state with less than 5% by weight of water.
Prince et al., Eur. J. of Epidemiology, Vol. 3, No. 2, Jun. 1987, discloses that a method of dry-heating a lyophilized factor VIII concentrate at 60.degree..degree.C. for 10 h results in a limited virus inactivation, but that hepatitis and also AIDS viruses can be transmitted by administering these dry-heated products. To increase the effectiveness of dry-heating, PCT application WO 88/08710 proposes a sequence of heat treatments.
Likewise, in EP-A-0 378 208, protein-containing compositons are subjected to a treatment with trialkyl phosphate in combination with a dry heating treatment.
The method of EP-B-0 159 311 suggests a treatment of blood products in the solid, wet state. A content of water, methanol or ethanol of more than 0.05 (5% by weight) and less than 0.70 (70% by weight) is adjusted and heating is effected in a closed container at a temperature in the range of from 50 .degree. to 121.degree. C.
EP-B-0 050 061 discloses a method comprising the treatment of biological and pharmaceutical products with from 0.25 to 10% by weight of a non denaturating amphiphile (detergent). In EP-B-0 131 740 it is, however, shown that the treatment with a detergent alone is relatively ineffective with regard to the virus inactivation. That publication suggests a mixture of a detergent and a di- or trialkylphosphate for an effective treatment. In that instance, the concentration of the detergent was 1% by weight and that of the solvent was 0.1% by weight .
It is to be understood that the use of the percentage symbol "%" refers to a percentage by weight.
A combined treatment with an organic solvent/detergent and with heat, wherein the blood product is heated in the dry state, has also been documented in the literature (American Journal of Hematology 35, 142 (1990).
Today, virus inactivation methods are termed as effective, if after application of the method on a blood product sample admixed with a high dose of a test virus (e.g., corresponding to a maximum possible titer of approximately 10.sup.5 in a coagulation factor preparation), it is no longer possible to detect any viruses, the virus titer thus having been reduced to below the detection limit.
As a measure of inactivation, the so-called reduction factor is known, which is calculated after a single addition of the test virus from the decadic logarithm of the quotient of the initial and final virus titers. From the directive EC III/8115/89-EN of the Commission of the European Communities, the so-called "total reduction factor" is known. It is calculated from the sum of the reduction factors of individual subsequent inactivation measures.
In modern medicine it is necessary to administer many blood products over long periods of time--in many cases even as a permanent treatment--in large quantities, also for prophylactic reasons. This will necessarily lead to a cumulation of infectious particles and thus to a substantially increased risk of infection, even if preparations that have already been virus-inactivated are administered.
Reduction factors must be compared with the so-called "worst case situation" for a virus contamination of the entire plasma pool. For example, it is known that plasma that tests negative for HIV content may yield an HIV content of up to 10.sup.5 ID/ml (infectious units per milliliter) in the plasma product. See Zeitschrift fur Allgemeine Medizin 65: 429-433 (1989). Thus, in the worst case, a seemingly virus-negative plasma product must be assumed to contain up to 10.sup.5 ID/ml. Accordingly, virus inactivation must take into account the worst case potential of virus contamination. For example, assuming that a patient is administered 100 l of a factor VIII preparation in the course of his life, a method of virus-inactivating plasma derivatives must thus permit a virus titer reduction of at least 10.sup.10 so as to avoid the infection of a patient with AIDS viruses.